HIV protease inhibitors, such as, ritonavir and saquinavir, are substrates for xenobiotic efflux pumps, e.g., P-glycoprotein and Mrp2 and thus penetrate the blood-brain barrier poorly. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice to validate signaling-based changes in blood-brain barrier transporter function in vivo. We recently found that blood-brain barrier P-glycoprotein transport activity could be reduced in vitro and in vivo by activating sphingolipid signaling, using sphingosine, sphingosine-1-phosphate (S1P) or fingolimod (FTY720 a prodrug currently in use to treat multiple sclerosis). To determine whether similar signaling to P-glycoprotein occurs in another intact tissue that is an important determinant of drug pharmacokinetics, we used an established comparative renal model, isolated killifish renal proximal tubules. This model permits direct measurements of P-glycoprotein transport activity. Isolated killifish tubules exposed to 0.01-1.0 &#956;M sphingosine-1-phosphate (S1P) exhibited a profound decrease in P-glycoprotein transport activity, measured as specific accumulation of a fluorescent cyclosporine A derivative in the tubule lumen. Loss of activity had a rapid onset and was fully reversible when the S1P was removed. S1P effects were blocked by a specific S1P receptor 1 (S1PR1) antagonist and mimicked by S1PR agonists, including FTY720. Sphingosine also reduced P-glycoprotein transport activity and those effects were blocked by an inhibitor of sphingosine kinase and by the S1PR1 antagonist. These results for a comparative renal model suggest that sphingolipid signaling to P-glycoprotein is not just restricted to the blood-brain and blood-spinal cord barriers, but occurs in other excretory and barrier tissues. We have been examining strategies to deliver therapeutic drugs that are P-glycoprotein substrates to the CNS. In an effort to inhibit the transporter, we designed dimers of the antipsychotic drug and P-gp substrate quetiapine (QT), linked by variable length tethers. In P-gp overexpressing cells and in human brain capillary endothelial hCMEC/D3 cells, the dimer with the shortest tether length (QT2C2) (1) was the most potent inhibitor showing >80-fold better inhibition of P-gp-mediated transport than monomeric QT. The dimers, which are linked via ester moieties, are designed to revert to the therapeutic monomer once inside the target cells. We demonstrated that the addition of two sterically blocking methyl groups to the linker (QT2C2Me2, 8) increased the half-life of the molecule in plasma 10-fold as compared to the dimer lacking methyl groups (QT2C2, 1), while retaining inhibitory potency for P-gp transport and sensitivity to cellular esterases. Experiments with purified P-gp demonstrated that QT2C2 (1) and QT2C2Me2 (8) interacted with both the H- and R-binding sites of the transporter with binding affinities 20- to 30-fold higher than that of monomeric QT. Using isolated rat brain capillaries, QT2C2Me2 (8) was a more potent inhibitor of P-gp transport than QT. Lastly, we showed that QT2C2Me2 (8) increased the accumulation of the P-gp substrate verapamil in rat brain in situ three times more than QT. Together, these results indicate that the QT dimer QT2C2Me2 (8) strongly inhibited P-gp transport activity in human brain capillary endothelial cells, in rat brain capillaries, and at the BBB in an animal model.